| With the rapid development of transgenic technology research,study of transgenic product detection technology has became an important part of global transgenic biosafety management.In past decades,PCR has been widely userid in transgenic detection.In recent years,nucleic acid isothermal amplification technology has developed rapidly.Compared with conventional PCR,nucleic acid isothermal amplification technology has the advantage that it does not depend on thermal cycling instrument,and can quickly amplify target fragments under constant temperature conditions.The isothermal technology is fast,simple and sensitive.Recombinase ploymerase amplification(RPA)is a nucleic acid isothermal amplification technology,in which high and low temperature cycle is not needed to achieve nucleic acid melting and annealing.Besides,droplet digital PCR(ddPCR)is also developed as a novel nucleic acid detection and quantification technology in recent years.The contents and results of this study are as follows:1.Detection of genetically modified maize Bt11(GM Bt11)by RPA and gel electrophoresis: according to the sequence of the joining region of GM Bt11 between exogenous phosphinothricin acetyl transferase gene(PAT)and vector backbone,primers were designed to achieve the construct-specific detection.Based on the RPA technology,a rapid and specific detection method of GM Bt11 is established.GM Bt11 could be detected at 37℃ in 20 mins.The results showed that the absolute and relative detection limit of RPA specific detection GM Bt11 were approximately 100 copies and 0.1%,respectively.2.Detection of exogenous nos terminator in transgenic maize by RPA and fluorescence detection: transgenic maize Bt11 was used in the study of the sensitivity of RPA fluorescence technology by detecting the exogenous nos terminator.The results showed that the absolute and relative detection limit were approximately 50 copies and 0.1%,respectively.As is known to all,the most commonly used quantitative method is Real-Time PCR.Nos terminator of transgenic maize Bt11 was also detected by fluorescent quantitative PCR dye method.It showed that the relative detection limit was approximately 0.1%,and the absolute detection limit was 10 copies or less.Detection of transgenic maize can be quantitatively detected by Real-Time PCR,however,the process is time-waste,and complicated quantitative standard curve is needed.By using RPA fluorescence technology,detetion time is shortened,within half an hour.The two technologies have their own advantages,and should be selected according to actual conditions.3.Determination of transgenic components of transgenic soybean GTS-40-3-2 by ddPCR: ddPCR and Real-Time PCR were used to determine 10% transgenic soybean GTS-40-3-2.The transgenic content were determined simultaneously by the two method,and the result turned out to be 10% which is the actual content.Compared with ddPCR,Real-Time PCR required highter amount of templates and primers,and more samples was required in standard curve plotting.Using the QX200 TM Droplet Digital TM PCR system,two sets of primers and probes were added to the same PCR tube,and thus the amount of templates is reduced.In data analysis process,standard curve is required according to the fluorescence value in Real-Time PCR,and then the gene copy number and the content of the transgenic component were obtained.In ddPCR,copy number of the target gene was obtained directly by the number of different fluorescent droplets without relying on the standard curve,and the content of transgenic components was calculated accurately.The results showed that the minimum detection limit of ddPCR was approximately 2 copies. |
